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received: 22 December 2015 accepted: 23 February 2016 Published: 15 March 2016
Pharyngeal pumping in Caenorhabditis elegans depends on tonic and phasic signaling from the nervous system Nicholas F. Trojanowski1,2,3, David M. Raizen1 & Christopher Fang-Yen2,3 Rhythmic movements are ubiquitous in animal locomotion, feeding, and circulatory systems. In some systems, the muscle itself generates rhythmic contractions. In others, rhythms are generated by the nervous system or by interactions between the nervous system and muscles. In the nematode Caenorhabditis elegans, feeding occurs via rhythmic contractions (pumping) of the pharynx, a neuromuscular feeding organ. Here, we use pharmacology, optogenetics, genetics, and electrophysiology to investigate the roles of the nervous system and muscle in generating pharyngeal pumping. Hyperpolarization of the nervous system using a histamine-gated chloride channel abolishes pumping, and optogenetic stimulation of pharyngeal muscle in these animals causes abnormal contractions, demonstrating that normal pumping requires nervous system function. In mutants that pump slowly due to defective nervous system function, tonic muscle stimulation causes rapid pumping, suggesting tonic neurotransmitter release may regulate pumping. However, tonic cholinergic motor neuron stimulation, but not tonic muscle stimulation, triggers pumps that electrophysiologically resemble typical rapid pumps. This suggests that pharyngeal cholinergic motor neurons are normally rhythmically, and not tonically active. These results demonstrate that the pharynx generates a myogenic rhythm in the presence of tonically released acetylcholine, and suggest that the pharyngeal nervous system entrains contraction rate and timing through phasic neurotransmitter release. Rhythmic muscle contractions are required for many aspects of physiology and behavior, from circulation to locomotion1. These rhythms can be described as myogenic, if intrinsic oscillations of membrane currents in the muscles drive contractions, or neurogenic, if a network of neurons acts as a central pattern generator (CPG) to drive muscle contraction. For example, vertebrate heart muscle generates its own rhythms. The autonomic nervous system modulates the rate and strength of cardiac contraction but does not provide any beat-to-beat timing information2, and innervation of the heart is dispensable for coordinated and effective cardiac function3. In contrast, a neural circuit in the vertebrate spinal cord controls locomotion by acting as a neural pacemaker, producing patterned activity that drives contraction of passively responding skeletal muscles4. Myogenic and neurogenic rhythms are not mutually exclusive: in some systems both the nervous system and muscles are capable of generating rhythms independently, and interact to generate rhythmic behavior. For example, leech heart motor neurons display rhythmic activity and entrain the myogenic rhythmic contractions of the heart5. Similarly, the crustacean pyloric dilator muscle exhibits a myogenic rhythm that is entrained by rhythmic activity in the stomatogastric neural system6. By contrast, mollusc heart motor neurons modulate heart rate over long time scales without entraining the heartbeat7. The mechanisms that underlie rhythmic behaviors have been most studied in invertebrates such as leeches, crustaceans, and molluscs due to the small number of neurons and relative ease of electrophysiological recordings in these animals. The ability to electrophysiologically identify the functional synaptic connectivity between neurons in these systems has enabled researchers to determine the roles of intrinsic and synaptic properties of 1
Department of Neurology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, 19104 PA, USA. 2Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, 19104 PA, USA. 3Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, Philadelphia, 19104 PA, USA. Correspondence and requests for materials should be addressed to D.M.R. (email:
[email protected]) or C.F.-Y. (email:
[email protected]) Scientific Reports | 6:22940 | DOI: 10.1038/srep22940
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Figure 1. The pharynx consists of three functional units, the corpus, isthmus, and terminal bulb. During a pump, food enters via the corpus. It is then transferred along the isthmus via posteriorly propagating peristaltic waves before being broken up by the cuticular grinder in the terminal bulb during the subsequent pump. Anterior is left.
individual neurons and muscles in generating rhythmic behaviors1,8,9. However, these organisms are not amenable to genetic approaches, limiting their utility for investigation of the genetic and molecular bases of rhythm generation in nervous systems and muscles. The nematode Caenorhabditis elegans represents a unique and powerful model for elucidating the genetic, neural, and muscular bases of behavior10,11. Among its strengths are its compact, extraordinarily well-mapped nervous system12,13, genetic manipulability, and optical transparency. The development of optical14,15 and electrophysiological16 methods for manipulating and monitoring neural activity has begun to enable analysis of the physiology and functional connectivity of C. elegans neural circuits. Such investigations apply a conceptual approach similar to that developed in leeches, crustaceans, and gastropods while leveraging the extensive genetic toolkit available in worms. Thus, C. elegans is well suited to provide insights into mechanisms that underlie rhythmic behaviors. C. elegans feeds on bacterial food via rhythmic contractions and relaxations of its pharynx (Fig. 1), a neuromuscular pump with similarities to the vertebrate and invertebrate heart. Like these hearts, the pharynx is a tube of electrically coupled muscle cells17–20 that pumps throughout the life of the animal. It responds to a variety of neurotransmitters and neuromodulators21, and relies on T- and L-type Ca2+ channels for its action potential22,23. The pharynx possesses a nervous system, a network of 20 neurons of 14 types, that is largely independent of the extra-pharyngeal nervous system and accounts for all chemical synapses onto pharyngeal muscle13. The role of the pharyngeal nervous system in the generation of rhythmic pharyngeal behavior is not yet clear. Laser ablation of all pharyngeal neurons does not completely abolish pharyngeal pumping24, nor does optogenetic hyperpolarization of all cholinergic pharyngeal motor neurons25,26, which normally excite pumping24,25,27. On the basis of these findings, the pharyngeal pumping rhythm has been described as myogenic24,28. However, pumping is abolished by genetic manipulations that eliminate cholinergic synaptic transmission29,30 or all synaptic transmission31–33, indicating that some nervous system function is required for pumping. Of the 20 pharyngeal neurons, the two cholinergic MC motor neurons appear to be the most important for regulation of rapid pumping: MC ablation dramatically decreases pump rate24,27, and optogenetic stimulation or inhibition of the MC neurons increases or decreases pump rate, respectively25. The MC neurons are activated by serotonin (5-HT)34 and appear to act primarily via a nicotinic acetylcholine (ACh) receptor containing the non-α subunit EAT-2, as eat-2 mutants resemble MC-ablated animals25,27,35,36. Electropharyngeograms (EPGs), extracellular recordings of pharyngeal muscle electrical activity37, reveal a very brief MC- and EAT-2-dependent depolarization preceding each muscle action potential during rapid pumping27. This depolarization may represent a response to pulsed neurotransmitter release from the MC neurons, but this idea is challenging to test since the activity patterns of the MC neurons are unknown and currently difficult to measure. We explored how the nervous system and pharyngeal muscle interact to control pumping, with the goal of comparing the mechanisms of pharyngeal contraction generation with those found in vertebrate and invertebrate hearts and other rhythmic systems. Our results demonstrate that the pharyngeal muscle generates a myogenic rhythm only in the presence of tonically released ACh, and suggest that the MC neurons stimulate pumping by rhythmically exciting and entraining the pharyngeal muscle rhythm in a manner similar to that by which the leech heartbeat is controlled by heart motor neurons.
Results
Pharyngeal pumping acutely requires nervous system function. The finding that pharyngeal pumping persists after laser ablation of the entire pharyngeal nervous system24 or after hyperpolarization of excitatory
Scientific Reports | 6:22940 | DOI: 10.1038/srep22940
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www.nature.com/scientificreports/ pharyngeal cholinergic neurons25,26, yet is abolished in mutants lacking ACh release29–33 suggests that ACh from the extra-pharyngeal nervous system is sufficient to induce feeding. However, since severe synaptic transmission mutations cause chronic changes in animal physiology and development, it is possible that the lack of feeding observed in these mutants may be explained by developmental abnormalities. To test the role of the nervous system in pumping while avoiding the confounding issue of abnormal development in mutant backgrounds, we sought to determine if the nervous system is acutely required for pumping. In order to acutely silence the nervous system, we expressed a histamine-gated chloride channel (HisCl) in all neurons using the Ptag-168 promoter38. HisCl activation has been shown to silence neurons in every case tested, both in C. elegans38,39 and in Drosophila40. Therefore, in worms expressing pan-neuronal HisCl, exogenous histamine is expected to lead to hyperpolarization of both pharyngeal and extra-pharyngeal neurons38, including excitatory cholinergic pharyngeal neurons such as the MCs. After 15 minutes on a 2% agarose pad containing 10 mM histamine, pumping completely ceased in worms expressing pan-neuronal HisCl (n > 80), while pumping persists on 2% agarose pads in worms lacking pan-neuronal HisCl41. Exposure of these worms to histamine does not dramatically affect pumping, as worms raised on histamine do not show severe growth defects38,39. Therefore, unlike the vertebrate, leech, and mollusc hearts, the pharynx requires a signal from the nervous system to produce myogenic contractions. Since ablation of the pharyngeal nervous system does not abolish pumping24, it appears that this signal can come from the extra-pharyngeal nervous system. However, since extra-pharyngeal neurons do not synapse on pharyngeal muscle, they cannot provide pump-to-pump timing information.
Normal pharyngeal muscle coordination requires the nervous system. We sought to better under-
stand the role of the pharyngeal nervous system in coordinating pharyngeal pumps. The pharynx can be divided into three functional units13 (Fig. 1). The anterior end of the pharynx contains the corpus, which draws in the bacterial food during muscle contraction. The posterior end of the pharynx contains the terminal bulb, which houses the grinder, three cuticular plates that crush the bacteria so their contents can be absorbed by the intestine. The isthmus connects the corpus and terminal bulb. Pharyngeal muscle fibers are oriented radially, so muscle contraction opens the lumen and relaxation closes it. During pumping, it is necessary for different parts of the pharynx to contract with slightly different timing to effectively transport food to the intestine42. A pharyngeal pump begins with the nearly simultaneous contraction of the corpus and the terminal bulb, drawing food particles into the pharyngeal lumen, followed by contraction of the anterior isthmus. After approximately 200 ms, these muscles begin to relax. The anterior tip of the corpus relaxes first, preventing food particles from escaping when the rest of the muscles relax43. Likewise, the corpus relaxes before the isthmus, allowing bacteria to be trapped in the anterior isthmus43,44. Posteriorly-propagating contractions of the posterior isthmus, known as peristalsis, transport bacteria from the anterior isthmus to the terminal bulb after about one out of every four pumps45. To gain insight into which aspects of pharyngeal pumping require the nervous system, we sought to determine the extent to which direct stimulation of pharyngeal muscle in the absence of nervous system function recapitulates normal muscle contraction patterns. In the absence of neural input, the vertebrate heart generates motions that are essentially the same as those observed with neural input; neural input modulates only the rate and force of cardiac contractions2. In contrast, in the leech heart, the electrical activity of the muscle is altered when the nervous system is hyperpolarized5. To test whether the pharynx produces motions in the absence of neural input that are similar to those produced in the presence of neural input, we silenced the nervous system using pan-neuronal HisCl activation and then stimulated the muscle directly using the light-activated excitatory opsin Chrimson expressed in pharyngeal muscle46. We used high-speed video recordings to examine the muscle contraction patterns of these worms in response to 200 ms optogenetic stimulation of pharyngeal muscle in the presence of histamine. We compared pan-neuronally hyperpolarized animals with muscle optogenetic stimulation to animals of the same strain without histamine or muscle stimulation, and we found two striking differences. First, in 31/37 experimental worms, we observed contraction in the terminal bulb but not in the corpus in response to optogenetic stimulation. The remaining 6/37 experimental worms showed feeble corpus contractions. By contrast, in the absence of histamine and optogenetic muscle stimulation, all worms of this strain had normal contractions of the corpus in addition to the terminal bulb (N = 15). The difference between the experimental and control worms is statistically significant (p
